Gotowa bibliografia na temat „Rifampicin‐resistant Mutants”

Antibiotic resistant mutants for rifampicin, streptomycin and klindamycin were isolated from the clinical isolate of Vibrio choleraeS mutated by chemical mutagens. Mutation frequency of V. cholerae S depends on the treatment time and the highest viable count of antibiotic resistant were for Rifampicin after treatment with Acridine orange, Ethedium bromide, Nitrosoguanidine, 5-Florouracil, 2-Bromouracil and cyclophosphamide. One thousand mutant isolates were examined for morphological differences in colony surface, color and diameter. The treatment with AO, NTG, 5-FU, and 2-BU gave opaque to orange color larger diameter about 5-7 mm of Rifampicin resistant mutant isolates at TSA and 25% of these mutant appeared as wrinkled surface. Klindamycin resistant mutants of V. cholerae S were appeared as similar to the wild type while, Streptomycin resistant mutants of appeared as pin- point white smooth colonies on TSA. No differences were seen for oxidase, string test and fermentation pattern for sucrose and lactose. Toxin CoregulatedPili production was differed from high level order designated as +++ to mild ++ and low level designated as + after mutation with 5-FU and 2-BU. However, 15% of rifampicin resistant mutant isolates gave no agglutination phenomena. No proteases activity detected even after 48 hour of incubation; the production of lipases enzyme did not affected; while, mutator isolates produced high level of β- haemolysins about 2.5 fold. About 90% of cyclophosphamide- rifampicin mutant showed homogenized culture with no auto agglutination but high level of proteases. While, only 10% of cyclophosphamide - rifampicin mutants gave slightly auto agglutination and didn’t produce proteases enzyme. The production of both TCP and CT were increased from rough pigment producing mutant isolates comparing with yellow and smooth mutants.

Aim. Streptomyces albus J1074 is one of the most popular streptomycete chassis for heterologous expression of natural product (NP) biosynthetic gene clusters (BGCs). There is keen interest in further improvement of the strain to provide increased yields of corresponding NPs. Introduction of certain types of antibiotic resistance mutations is a proven way to improve Streptomyces strains. For example, selection for increased resistance to rifampicin is known to lead to increased antibiotic activity. Here we used available lineages of antibiotic-resistant mutants of S. albus to raise rifampicin-resistant variants (Rifr) and to study their properties. Methods. Microbiological and molecular genetic approaches were combined to generate Rifr mutants and to study their properties. Results. By plating S. albus onto GYM agar supplemented with 10 mcg/mL of rifampicin, we isolated 85 stable Rifr colonies, whose resistance level was within 10-200 mcg/mL range. Sequencing revealed wide spectrum of missense mutations within rpoB gene. Bioassays demonstrated dramatically increased endogenous antibiotic activity of certain Rifr mutants. Conclusions. Selection for rifampicin resistance is a viable way to increase the yields of NPs in S. albus. Keywords: Streptomyces albus J1074, antibiotic resistance, rifampicin.

SUMMARYThe antibiotic resistance ofMycoplasma mycoidesssp.mycoidesstrain T1was investigated. This strain was resistant to high levels ( > 100 μg ml−1) of rifampicin and nalidixic acid. It was sensitive to streptomycin, spectinomycin and novobiocin; however, single step mutants with high levels of resistance ( > 100 μg ml−1) were readily isolated. With erythromycin and tylosin for which the minimum inhibitory concentration (MIC) for the parent strain was < 0·1 μg ml−1, mutants resistant to > 100 μg ml−1were obtained in two and three steps respectively. The MIC of tetracycline in single step resistant mutants (0·6 μg ml−1) was tenfold higher than the parent strain, but could not be increased further. There was only a twofold increase in resistance to chloramphenicol in single step mutants. The frequency of resistant mutants varied with the antibiotic and was between 4× 10minuss;6and 2× 10−8. The mutation rate to antibiotic resistance to streptomycin, spectinomycin, novobiocin, erythromycin and tylosin was between 3× 10−8and 5× 10−9per cell per generation. There was a fivefold decrease in mutation rate to resistance to 60 μg ml−1streptomycin compared to that to 20 μg ml−1.

Rifampicin is a critical first-line antibiotic for treating mycobacterial infections such as tuberculosis, one of the most serious infectious diseases worldwide. Rifampicin resistance in mycobacteria is mainly caused by mutations in the rpoB gene; however, some rifampicin-resistant strains showed no rpoB mutations. Therefore, alternative mechanisms must explain this resistance in mycobacteria. In this work, a library of 11,000 Mycobacterium smegmatis mc2 155 insertion mutants was explored to search and characterize new rifampicin-resistance determinants. A transposon insertion in the MSMEG_1945 gene modified the growth rate, pH homeostasis and membrane potential in M. smegmatis, producing rifampicin resistance and collateral susceptibility to other antitubercular drugs such as isoniazid, ethionamide and aminoglycosides. Our data suggest that the M. smegmatis MSMEG_1945 protein is an ion channel, dubbed MchK, essential for maintaining the cellular ionic balance and membrane potential, modulating susceptibility to antimycobacterial agents. The functions of this new gene point once again to potassium homeostasis impairment as a proxy to resistance to rifampicin. This study increases the known repertoire of mycobacterial ion channels involved in drug susceptibility/resistance to antimycobacterial drugs and suggests novel intervention opportunities, highlighting ion channels as druggable pathways.

Background: Multidrug-resistant strains of Acinetobacter baumannii have been reported increasingly around the world. The administration of an association of antibiotics has been proposed to create an active combination and to prevent the emergence of resistance. Methodology: The activity of colistin, rifampicin, gentamicin, imipenem and their associations was evaluated by means of killing curves in fourteen isolates belonging to three endemic PFGE types, in a university hospital of Buenos Aires city. The 14 isolates were selected on the basis of different mechanisms responsible for resistance to carbapenems and different susceptibility to colistin. Results: The mechanism responsible for the resistance to imipenem was the production of OXA-23 and OXA-58 carbapenemases. Heteroresistance to colistin was observed in six isolates. The associations colistin-rifampicin and colistin-imipenem were synergistic in heteroresistant isolates and prevented the development of colistin-resistant mutants. The association imipenem-gentamicin was bactericidal in gentamicin susceptible isolates, whereas the association imipenem-rifampicin was always indifferent. Conclusion: The antimicrobial activity and the presence of synergy are related to the antimicrobials' susceptibilities irrespective of the PFGE type or the OXA-carbapenemase produced.

Rhodothermus marinus is a halophilic extreme thermophile, with potential as a model organism for studies of the structural basis of antibiotic resistance. In order to facilitate genetic studies of this organism, we have surveyed the antibiotic sensitivity spectrum of R. marinus and identified spontaneous antibiotic-resistant mutants. R. marinus is naturally insensitive to aminoglycosides, aminocylitols and tuberactinomycins that target the 30S ribosomal subunit, but is sensitive to all 50S ribosomal subunit-targeting antibiotics examined, including macrolides, lincosamides, streptogramin B, chloramphenicol, and thiostrepton. It is also sensitive to kirromycin and fusidic acid, which target protein synthesis factors. It is sensitive to rifampicin (RNA polymerase inhibitor) and to the fluoroquinolones ofloxacin and ciprofloxacin (DNA gyrase inhibitors), but insensitive to nalidixic acid. Drug-resistant mutants were identified using rifampicin, thiostrepton, erythromycin, spiramycin, tylosin, lincomycin, and chloramphenicol. The majority of these were found to have mutations that are similar or identical to those previously found in other species, while several novel mutations were identified. This study provides potential selectable markers for genetic manipulations and demonstrates the feasibility of using R. marinus as a model system for studies of ribosome and RNA polymerase structure, function, and evolution.

Thesis (MScMedSc)--Stellenbosch University, 2013.
ENGLISH ABSTRACT: Multidrug resistant tuberculosis (MDR-TB), defined as having resistance to at least the first-line drugs, isoniazid and rifampicin (RIF), is a global health problem. Mutations in the rpoB gene, encoding the β-subunit of RNA polymerase, are implicated in RIF resistance - with the S531L and H526Y mutations occurring most frequently. The level of RIF resistance varies for strains with identical rpoB mutations, which suggests that other factors play a role in RIF resistance. Efflux has been implicated in determining the intrinsic level of RIF resistance. Increased expression of the multidrug efflux pump, Rv1258c, following RIF exposure was observed in some Mycobacterium tuberculosis MDR clinical isolates and H37Rv RIF mono-resistant mutants, but not others. The factors influencing the induction of Rv1258c are poorly understood. The aim of this study was to investigate the effects of rpoB mutations on expression of Rv1258c and whiB7, a transcriptional regulator of Rv1258c, in M. tuberculosis H37Rv in vitro generated RIF resistant mutants, in the absence and presence of RIF. The promoter region of M. tuberculosis H37Rv Rv1258c was cloned into a position upstream of a lacZ gene (encoding β-galactosidase) in multi-copy episomal and integrating vectors. Vector functioning and the effect of rpoB mutations on Rv1258c promoter activity were initially investigated in the non-pathogenic related species, Mycobacterium smegmatis mc2155 rpoB mutants and subsequently in M. tuberculosis by doing β-galactosidase assays. qRT-PCR was done to investigate the effects of rpoB mutations on native Rv1258c and whiB7 gene expression. Episomal and integrating vectors were functional and the integrating vector system was used for subsequent β-galactosidase assays in M. tuberculosis. Rv1258c promoter activity in the S531L mutant was approximately 1.5 times less and in the H526Y mutant 1.5 times higher than that of the wild-type in M. smegmatis. Similarly, Rv1258c promoter activity in the S531L mutant was approximately half and in the H526Y mutant approximately double that of the wild-type in M. tuberculosis. A similar trend in Rv1258c and whiB7 expression to those observed using β-galactosidase assays were observed when investigating the native Rv1258c and whiB7 gene transcript levels compared to the wild-type using qRT-PCR, although differences were not significant. Exposure of the M. smegmatis and M. tuberculosis rpoB mutants to sub-inhibitory levels of RIF did not affect Rv1258c promoter activity. Mutations in rpoB had a marginal effect on Rv1258c and whiB7 transcript levels, but showed the same trend as that seen for Rv1258c promoter activity. It remains to be determined whether these differences are biologically significant. When considering efflux pumps as new targets for treatment, possible differences in efflux pumps expression due to different rpoB mutations should be considered.
AFRIKAANSE OPSOMMING: Multi-middel weerstandige tuberkulose (MDR-TB) word gedefinieer as weerstandigheid tot ten minste rifampisien (RIF) en isoniasied, wat deel van die eerstelyn anti-tuberkulose behandeling vorm. Mutasies in die rpoB geen, wat die β-subeenheid van die RNA polimerase enkodeer, word geassosieer met RIF weerstandigheid. S531L en H526Y rpoB mutasies kom die algemeenste voor. RIF weerstandigheids vlakke verskil egter tussen isolate met identiese rpoB mutasies, wat impliseer dat ander faktore ook 'n rol in RIF weerstandigheid speel. 'n Toename in transkripsie van die Rv1258c geen, wat 'n multi-middel effluks pomp enkodeer, is waargeneem met blootstelling aan RIF, slegs in sommige M. tuberculosis H37Rv RIF mono-weerstandige mutante and MDR kliniese isolate, maar nie in ander nie. Die faktore wat die induksie van die Rv1258c effluks pomp beïnvloed is nie goed nagevors nie. Die studie ondersoek die effek van die rpoB mutasies op die uitdrukking van die Rv1258c en whiB7,'n transkripsionele regulator van Rv1258c, gene in M. tuberculosis H37Rv in vitro gegenereerde RIF weerstandige mutante, in die teenwoordigheid en afwesigheid van RIF. Die promotor area van die M. tuberculosis H37Rv Rv1258c geen is in 'n posisie stroomop van 'n lacZ geen, wat vir β-galaktosidase enkodeer, in multi-kopie episomale en integreerende vektors ingekloneer. Die funksionaliteit van die vektor en effek van rpoB mutasies op Rv1258c promotor aktiwiteit is ondersoek in die naverwante nie-patogeniese spesies, M. smegmatis en daarna in M. tuberculosis deur β-galaktosidase essais te doen. qRT-PCR is gedoen om die effek van rpoB mutasies op die vlak van transkripsie van die natuurlike Rv1258c geen en die whiB7 geen te bestudeer. Beide die episomale en integreerende vektors was funksioneel en daar is besluit om die integreerende vektor vir daaropeenvolgende β-galaktosidase essais in M. tuberculosis te gebruik. Rv1258c promotor aktiwiteit van die S531L mutant was ongeveer 1.5 keer minder as en die van die H526Y mutant 1.5 keer hoër as die van die ongemuteerde bakterië in M. smegmatis. Soortgelyk was die Rv1258c promoter aktiwiteit van die S531L mutant ongeveer die helfde van en die van H526Y mutant ongeveer dubbel die van die ongemuteerde bakterië in M. tuberculosis 'n Soortgelyke neiging in die vlakke van Rv1258c en whiB7 transkripte van die natuurlike geen is gedurende qRT-PCR waargeneem alhoewel die verskille nie beduidend was nie. Blootstelling aan sub-inhibitoriese konsentrasies van RIF het geen effek op Rv1258c uitdrukking in die M. smegmatis of M. tuberculosis rpoB mutante gehad nie. Die rpoB mutasies het net 'n effense effek op Rv1258c en whiB7 transkrip vlakke in M. tuberculosis rpoB mutante, maar transkrip vlakke het 'n soortgelyke neiging as die Rv1258c promoter aktiwiteit getoon. Of die waargenome verskille biologies betekenisvol is, moet nog bepaal word. Indien effluks pompe as teikens vir bahandeling gebruik sou word, moet in ag geneem word dat effluks pompe moontlik verskillend uitgedruk word in verskillende rpoB mutante.
The DST/NRF Centre of Excellence in Biomedical Tuberculosis Research, Stellenbosch University
DAAD-NRF in Country Scholarship and Ernst and Ethel Eriksen Trust
Harry Crossley Foundation

Bacterial persisters are a subpopulation of bacteria that can tolerate lethal concentrations of antibiotics. These are phenotypic variants that can give rise to drug‐susceptible population upon withdrawal of the antibiotic. Persistent bacteria play a crucial role in prolonging antibiotic treatment and are responsible for the recalcitrance of many chronic bacterial diseases, including tuberculosis. Several mechanisms have been proposed for the formation of persisters, which include expression of toxin‐antitoxin systems, generation of reactive oxygen species (ROS), and stochastic changes in gene expression and so on. Recent report from our laboratory has demonstrated that continuous prolonged exposure of Mycobacterium tuberculosis cells to lethal concentrations of antibiotics generates antibiotic persistence phase cells from which genetically resistant mutants emerge de novo either to the same antibiotic to which it was exposed to or to another antibiotic used for selection Sebastian et al., Antimicrobial Agents Chemotherapy 61(2), e01343‐16, 2016). The persistence phase cells showed high levels of oxidative stress that inflicted genome‐wide mutations in addition to the mutations for which the resistant mutants were selected against rifampicin and moxifloxacin. Thus, it was we demonstrated that the antibiotic persistence phase M. tuberculosis cells is a reservoir for the de novo emergence of antibiotic resistant mutants. In the present study, the response of Mycobacterium smegmatis upon prolonged exposure to rifampicin was examined since the bacilli has two mechanisms to inactivate or neutralise the action of rifampicin. These mechanisms include: (i). ADP‐ribosylation of rifampicin by the product of the gene ADP‐ribosyltransferase (arr); (ii). Rescue of rifampicin‐ mediated transcription inhibition by MsRbpA. The question asked was whether genetically resistant mutants against rifampicin would emerge from rifampicin persister phase cells, like in the case of M. tuberculosis cells and if they do, what are the mechanisms by which the rifampicin‐resistant mutants emerge from the persistence phase cells. For comparison and contrast purpose, and as a control sample, the response of M. smegmatis cells to moxifloxacin, against which the bacilli do not have any inherent inactivation or neutralisation mechanism, was studied. The Chapter 1, which forms the Introduction to the thesis, gives an extensive literature survey on all the different aspects of the research performed on the response of mycobacterial cells to antibiotics. The Chapter 2 presents in detail all the materials and methods used to perform the experiments. A large number of cell biological and molecular biological methods, such as fluorescence microscopy and fluorescence measurements, flow cytometry, cloning and expression, real time RT‐PCR and whole genome sequencing, and biophysical methods such as electron paramagnetic resonance spectrometry, and others were used to perform the experiments. The data Chapter 3 presents the data on the response of M. smegmatis cells to rifampicin. The data shows that exposure to MBC levels of rifampicin results in the killing of the cells to a 5‐log10 reduction in the cfu of M. smegmatis cells but the remaining cells persist and from these cells emerge rifampicin‐resistant mutants. The persistence phase cells were found to generate elevated levels of hydroxyl radical, which inflicted genome‐wide mutations, and the mutants harbouring nucleotide changes at the rifampicin resistance determining region (RRDR) could regrow back. Interestingly, the killing phase and the regrowth phase showed very low levels of hydroxyl radical unlike the persistence phase cells. The mutations, which are identical to those in the rifampicin‐resistant mutants have been reported in the M. tuberculosis cells isolated from in vitro cultures and from the TB patients. The data Chapter 4 presents the response of the arr knockout mutant to rifampicin. The persistence phase population of the arr knockout mutant showed significantly higher levels of hydroxyl radical generation than the equivalent persistence phase population of the wild type cells. While the wild type cells showed emergence of rifampicin‐resistant mutants from the persistence phase, the arr knockout mutant showed the emergence of rifampicin‐ resistant mutants from the very exposure of the cells to rifampicin. In other words, the natural mutation frequency of the arr knockout mutant was significantly higher than that of the wild type. This indicated that the arr gene might have a natural role in keeping the oxidative stress at lower levels in the cells, which needs further investigation. The data Chapter 5 presents the response of M. smegmatis cells to moxifloxacin. Here also, the bacilli exposed to lethal concentrations of moxifloxacin showed a killing phase, followed by a persistence phase and a regrowth phase. The moxifloxacin‐resistant mutants were found to emerge from the moxifloxacin persistence phase cells. The cells from the regrowth phase of moxifloxacin‐exposed cells showed mutations in the quinolone resistance determining region (QRDR) in the gyrase gene, which is the target of moxifloxacin. The mutations, which are identical to those in the rifampicin‐resistant mutants have been reported in the M. tuberculosis cells isolated from in vitro cultures and from the TB patients. The thesis is concluded with discussion of the findings presented in the three chapters by projecting the comparison and contrast of the response of M. smegmatis and M. tuberculosis cells to rifampicin. The thesis contains an extensive bibliography.